Expression of glucose transporter-2 in murine retina: Evidence for glucose transport from horizontal cells to photoreceptor synapses.

The retina has the highest relative energy consumption of any tissue, depending on a steady supply of glucose from the bloodstream. Glucose uptake is mediated by specific transporters whose regulation and expression are critical for the pathogenesis of many diseases, including diabetes and diabetic retinopathy. Here, we used immunofluorescence to show that glucose transporter-2 (GLUT2) is expressed in horizontal cells of the mouse neuroretina in proximity to inner retinal capillaries. To study the function of GLUT2 in the murine retina, we used organotypic retinal explants, cultivated under entirely controlled, serum-free conditions and exposed them to streptozotocin, a cytotoxic drug transported exclusively by GLUT2. Contrary to our expectations, streptozotocin did not measurably affect horizontal cell viability, while it ablated rod and cone photoreceptors in a concentration-dependent manner. Staining for poly-ADP-ribose (PAR) indicated that the detrimental effect of streptozotocin on photoreceptors may be associated with DNA damage. The negative effect of streptozotocin on the viability of rod photoreceptors was counteracted by co-administration of either the inhibitor of connexin-formed hemi-channels meclofenamic acid or the blocker of clathrin-mediated endocytosis dynasore. Remarkably, cone photoreceptors were not protected from streptozotocin-induced degeneration by neither of the two drugs. Overall, these data suggest the existence of a GLUT2-dependent glucose transport shuttle, from horizontal cells into photoreceptor synapses. Moreover, our study points at different glucose uptake mechanisms in rod and cone photoreceptors.

Clusters of neuronal neurofascin prefigure the position of a subset of nodes of Ranvier along individual central nervous system axons in vivo.

The spacing of nodes of Ranvier crucially affects conduction properties along myelinated axons. It is assumed that node position is primarily driven by growing myelin sheaths. Here, we reveal an additional mechanism of node positioning that is driven by the axon. Through longitudinal live imaging of node formation dynamics in the zebrafish central nervous system, we show that stable clusters of the cell adhesion molecule neurofascin a can accumulate at specific sites along axons prior to myelination. While some of these clusters are pushed into future node position by extending myelin sheaths, others are not and thus prefigure the position of where a mature node forms. Animals that lack full-length neurofascin a show increased internodal distances and less regular nodal spacing along single axons. Together, our data reveal the existence of an axonal mechanism to position nodes of Ranvier that does not depend on regulation by myelin sheath growth.

Visualization and Time-Lapse Microscopy of Myelinating Glia In Vivo in Zebrafish.

In vivo time-lapse microscopy provides important information about the kinetics of cellular events and their control by interactions with neighboring cells. Here, we describe the generation and use of transgenic zebrafish to visualize dynamics of myelinating glia using cell type-specific expression and microscopy of genetically encoded fluorescent proteins. With this method, we are able to simultaneously separate and trace up to three different colors over time.

Evidence for Myelin Sheath Remodeling in the CNS Revealed by In Vivo Imaging.

The length of myelin sheaths affects conduction speed along axons and information propagation. It has recently become clear that myelin may be adaptively modified to modulate circuit function, implying that length remodeling of myelin sheaths should occur. However, direct evidence for such events is lacking. We have investigated how myelination patterns are formed, maintained, and remodeled using long-term imaging and myelin ablation in zebrafish. We demonstrate that length differences between myelin sheaths are established by rapid and variable growth within 3 days after their formation, independently of their time of formation, and even along discontinuously myelinated axons. Afterward, sheaths continue extending at similar rates to compensate for overall animal growth. In consequence, once axon myelination patterns are established, they are maintained over long periods of time. We tested whether mature myelin sheaths can remodel by removing individual sheaths from single axons by targeted ablation. Remarkably, extensive changes in sheath length and number occurred, which frequently restored the original myelination pattern. Our results show that axons can control myelin growth and remodeling, and we provide evidence for a homeostatic control of axon myelination patterns by maintenance and remodeling of myelin sheath length, with implications for circuit development, function, and repair.